Selectivity of a C-terminal peptide antiserum for different DNA-binding states of the vitamin D receptor

Archives of Biochemistry and Biophysics
N J KoszewskiR L Horst

Abstract

Antisera against peptides from the extreme N- and C-terminal regions of the VDR were evaluated. The N-terminal antiserum Ab192 functioned as a general-purpose antibody, being able to supershift the rhVDR in heterodimeric or homodimeric binding complexes in the EMSA, and detect both recombinant and native forms of the receptor on Western blots. The C-terminal antiserum, Ab195, also identified the receptor on Western blots, but in contrast, it displayed differential sensitivity to the conditions employed in the EMSA. In the presence of 1,25(OH)2D3, rhVDR, rhRXR alpha, and nonspecific DNA, Ab195 produced a weak supershift of the heterodimer complex in the EMSA. Significantly, omission of hormone from the binding buffer resulted in a complete shift of the bound complex with the antiserum. A complete supershift was also observed if only the nonspecific DNA was removed. Together these results indicate antiserum sensitivity to the ligand status in the rhVDR C-terminus as part of a DNA-bound heterodimer complex. Inclusion of 9-cis RA resulted in a modest increase in the amount of shifted product relative to 1,25(OH)2D3 alone. Finally, Ab195 completely supershifted the rhVDR homodimer binding complex under all tested conditions, includi...Continue Reading

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Citations

May 11, 1999·Journal of Bone and Mineral Research : the Official Journal of the American Society for Bone and Mineral Research·N J KoszewskiR L Horst
Nov 26, 1999·Journal of Bone and Mineral Research : the Official Journal of the American Society for Bone and Mineral Research·J RussellN J Koszewski
Apr 28, 2010·The Journal of Steroid Biochemistry and Molecular Biology·Nick J KoszewskiHartmut H Malluche
Oct 13, 2000·American Journal of Physiology. Cell Physiology·T A ReinhardtR L Horst
Jun 6, 2003·Steroids·Nicholas J Koszewski, Anne Rowan
Apr 20, 2001·Neuroscience·M C LangubN J Koszewski
Apr 25, 2000·The Journal of Steroid Biochemistry and Molecular Biology·N J KoszewskiJ Russell

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